Bioactive compound modulation of epigenetic regulator Sp1/NFkB/miR network in AML

描述（由申请人提供）：尽管在了解髓性白血病发生的生物学机制方面取得了进展，但急性髓性白血病（AML）仍然是大多数患者的致命疾病。文献表明，异常DNA甲基转移酶（DNMT）依赖的DNA超甲基化介导的肿瘤抑制基因（TSGs）沉默在AML的发生和进展中起着关键的致病作用。氮杂核苷类药物，即地西他滨，最近已被FDA批准作为低甲基化药物，并在一些患者中取得了积极的临床结果。然而，临床反应仅限于少数造血恶性肿瘤。因此，迫切需要进一步的研究来探索新的治疗策略或药物来克服令人沮丧的结果。我们的长期目标是阐明控制DNA甲基化的调控机制，从而将白血病的发生作为开发可用于减轻疾病过程的治疗方案的先决条件。这项拨款申请的目的是通过地西他滨的不同机制，探索具有不同结构的新型DNA低甲基化剂（DNMTi）。本研究提出的具体假设是，生物活性食品成分百里醌（Thymoquinone， TQ）可能通过调节甲基化调控因子Sp1/NFkB/miR网络实现更高的DNA低甲基化功效。该假设基于以下观察结果：1)miR29b直接破坏DNMT3a/3b，并通过损害其反激活子Sp1间接消除DNMT1，从而导致DNA低甲基化。2) Sp1/NFkB复合物抑制miR29b的表达，并与AML细胞系和患者样本中的DNMT水平呈正相关，表明DNA甲基化受一个涉及NFkB活性、Sp1/NFkB复合物、DNMT和miRs的蛋白- mir网络的控制。3)植物源性药物显示出有效的抗白血病活性，具有巨大的治疗潜力，而传统药物存在许多副作用；4)TQ是一种生物活性成分，通过显著阻断NFkB信号通路，具有抗癌作用，对正常细胞的毒性很小。基于这些观察结果，本提案的实验重点是TQ在体外和体内的低甲基化作用。具体目的是提供对TQ作用机制的全面了解，并优化TQ对白血病有效的剂量和给药方案。此应用程序的具体目标是：1。通过证明TQ通过Sp1/NFkB/miR29b网络在AML细胞系和患者原代细胞中作为DNA低甲基化剂起作用，阐明TQ抗白血病活性的机制。我们将通过i) Western blot和定量PCR (qPCR), ii) LC/MS/MS和iii) MTS和PI/AV染色，证明TQ对Sp1/NFkB/miR29b调控网络的药理学修饰可诱导体外DNA低甲基化。2. 对TQ在白血病小鼠模型中的药效学和药代动力学活性进行临床前体内评估。我们将使用Western blot、qPCR和LC/MS/MS确定TQ给药的有效药理学剂量和时间表，以调节体内失衡的Sp1/NFkB/miR29b网络，从而实现体内DNA低甲基化；ii)确定TQ的血浆和细胞内PK参数，并将其与PD和临床疗效终点相关联；iii)确定通过生存时间测量的临床疗效。该项目将由具有翻译研究专业知识（Liu）和PK/PD研究专业知识（Chan）的研究人员通过跨学科方法进行。如果成功，这种新型的DNMTi可以应用于实体肿瘤或非增生性恶性肿瘤，这项研究可能会促进我们对特定生物活性食物成分介导的表观遗传变化的理解，Sp1/NFkB、miR和DNA甲基化在白血病发生中的作用，TQ作用的分子途径，并有助于我们充分评估个体营养物质的特异性。

DESCRIPTION (provided by applicant): Despite progress made in understanding the biological mechanisms of myeloid leukemogenesis, acute myeloid leukemia (AML) remains a deadly disease for most of the patients. It is well documented that silencing of tumor suppressor genes (TSGs) mediated by aberrant DNA methyltransferase (DNMT)-dependent DNA hypermethylation plays a critical pathogenic role in the development and progression of AML. Azanucleosides, i.e., decitabine, have been recently approved by FDA as hypomethylating agents and positive clinical outcome has been achieved for some patients. However, the clinical response is restricted to a minority of hematopoietic malignancies. Hence, further studies are urgently required to explore novel therapeutic strategies or agents to overcome the dismal outcome. Our long-term goals are to elucidate the regulatory mechanisms controlling DNA methylation thereby leukemogenesis as a prerequisite to the development of therapeutic protocols that can be used to attenuate the disease process. The objectives of this grant application are to explore novel DNA hypomethylation agents (DNMTi) with diverse structures through distinct mechanisms from decitabine. The specific hypothesis behind the proposed research is that bioactive food component Thymoquinone (TQ) may achieve higher efficacy of DNA hypomethylation through modulating methylation regulator Sp1/NFkB/miR network. That hypothesis is based on the following observations: 1) miR29b directly disrupts DNMT3a/3b and indirectly abolishes DNMT1 via impairment of its transactivator Sp1, thereby leading to DNA hypomethylation. 2) Sp1/NFkB complex suppresses miR29b expression and positively correlates to DNMT level in AML cell lines and patient samples, suggesting that DNA methylation is under control of a protein-miR network involving NFkB activity, Sp1/NFkB complex, DNMTs and miRs. 3) plant-derived drugs displayed efficiently anti-leukemic activities with huge therapeutic potentials, while the conventional medicine has lots of side effects, 4) TQ is a bioactive constituent and acts as anticancer agent, with minimal level of toxicity to normal cells, by significantly blocking NFkB signaling pathways. Based on these observations, the experimental focus of this proposal is on the hypomethylating effect of TQ in vitro and in vivo. The specific aims are designed to provide a comprehensive understanding of the mechanism(s) of TQ action(s) and to optimize the dose and schedule of administration of TQ effective against leukemic disease. The specific aims of this application are to: 1. Elucidate the mechanism of TQ antileukemic activity by the demonstration that TQ functions as DNA hypomethylation agent through Sp1/NFkB/miR29b network in AML cell lines and patient primary blasts. We will demonstrate that pharmacological modification of Sp1/NFkB/miR29b regulatory network by TQ induces DNA hypomethylation in vitro using i) Western blot and quantitative PCR (qPCR), ii) LC/MS/MS and iii) MTS and PI/AV staining. 2. Perform preclinical in vivo evaluation of the pharmacodynamic and pharmacokinetic activity of TQ in leukemic mice models. We will i) define the effective pharmacological dose and schedule of TQ administration that will modulate the misbalanced Sp1/NFkB/miR29b network thereby DNA hypomethylation in vivo using Western blot, qPCR, and LC/MS/MS, ii) determine the plasma and intracellular PK parameters of TQ and correlate these with PD and clinical efficacy endpoints, iii) determine the clinical efficacy measured by survival duration. This project will be carried out through an interdisciplinary approach by investigators with expertise in translational research (Liu) and PK/PD studies (Chan). If successful, the novel DNMTi can be applied to solid tumor or non-proliferating malignancies, this investigation may advance our understanding of epigenetic changes mediated by specific bioactive food component, the roles of Sp1/NFkB, miR and DNA methylation in leukemogenesis, the molecular pathways of TQ action and help us to adequately evaluate the specificity of individual nutrient.